CN114277023B

CN114277023B - Recombinant nitrile hydratase and application thereof in preparation of nicotinamide by coupling ion exchange resin

Info

Publication number: CN114277023B
Application number: CN202111633257.7A
Authority: CN
Inventors: 郑建永; 王祎丁; 宋问; 孙杰; 于欣君
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2021-12-29
Filing date: 2021-12-29
Publication date: 2024-03-26
Anticipated expiration: 2041-12-29
Also published as: CN114277023A

Abstract

The invention provides a recombinant nitrile hydratase and application thereof in preparing nicotinamide by coupling ion exchange resin, wherein concentrated solution of supernatant obtained by fermenting and culturing wet bacterial cells obtained by fermenting and culturing recombinant nitrile hydratase gene engineering bacteria is taken as a catalyst, 3-cyanopyridine is taken as a substrate, the ion exchange resin is added, phosphoric acid buffer solution with pH value of 6-9 is taken as a reaction medium to form a reaction system, hydration reaction is carried out at 20-30 ℃ and 100-1000rpm, and after the reaction is completed, reaction solution is separated and purified to obtain the product nicotinamide. The recombinant nitrile hydratase coupling ion exchange resin has the advantages of mild condition, high efficiency, high chemical selectivity, high regioselectivity and the like, and the biocatalysis process has the characteristics of no toxicity, no pollution, low energy consumption and the like, is an environment-friendly synthesis method, adsorbs more than 95 percent of nicotinic acid impurities, and obtains nicotinamide products with the purity of more than 99.99 percent.

Description

Recombinant nitrile hydratase and application thereof in preparation of nicotinamide by coupling ion exchange resin

Technical Field

The invention belongs to the field of biochemical engineering, and relates to a recombinant nitrile hydratase and application of the recombinant nitrile hydratase in preparing high-purity nicotinamide by catalyzing and hydrating 3-cyanopyridine coupling ion exchange resin.

Background

Nicotinamide (trade name nicotinamide), a small molecule vitamin that is water-soluble, stable, and readily penetrates the stratum corneum. It has high safety as a pharmaceutical ingredient and is also a basic vitamin supplement in clinical dermatological treatment. In recent years, the research proves that the nicotinamide as an efficacy component of the cosmetics has good effects of inhibiting melanin deposition, resisting inflammation and aging, treating acne and the like. Generally, the amount of niacinamide added is more than 3% to exert the whitening effect, and a high content of niacinamide may cause a certain irritation to skin, which may be caused by a small amount of niacin in niacinamide. Nicotinamide can decompose under acidic condition to generate nicotinic acid, which causes skin to appear reddening, itching, stinging and the like, namely intolerance phenomenon.

Conventional amide production processes can be broadly divided into three categories: condensing carboxylic acid with amine substances; hydrolysis of nitriles; and amidation of thioacids with azides. The chemical synthesis method needs severe reaction conditions of high temperature and high pressure of 200-400 ℃, has long reaction process, needs to add metal catalysts such as framework copper and the like, is accompanied with the generation of toxic byproducts in the reaction process, and is difficult to meet the current green chemistry requirements. Compared with chemical synthesis, the biocatalytic synthesis of the amide has the advantages of mild reaction conditions, no toxic by-product generation, excellent regio-selectivity, stereoselectivity and the like.

Nitrile hydratase (EC 4.2.1.84) is an enzyme that catalyzes the conversion of nitriles to the corresponding amides. The nitrile hydratase uses a sulfur atom and a cysteine-sulfinic acid residue as active centers, and the minimum functional unit is generally composed of two subunits (α and β subunits) and one metal ion, and the nitrile hydratase is classified into Co-type nitrile hydratase and Fe-type nitrile hydratase depending on the metal ion chelated thereto. Nitrile hydratase is a biocatalyst with high catalytic efficiency and strong specificity (including substrate specificity, stereoisomer specificity and chemical specificity). However, because of the characteristics of the nitrile hydratase gene cluster, the characteristics of partial nitrilase and the protein characteristics of the enzyme, all factors which can cause protein denaturation can cause the inactivation of the enzyme, such as pH, heavy metal salt, heat, ultraviolet rays, violent vibration and the like. Even under the optimal catalytic conditions, the enzyme catalytic reaction rate gradually decreases with the extension of the reaction time. In addition, the free enzyme produces impurities after the catalytic hydration hydrolysis reaction, which makes the separation and purification process of the product difficult, resulting in an increase in production cost. These factors have greatly limited the use of nitrile hydratase catalysts in modern industries. Therefore, the nicotinamide synthesis process and related substance research are carried out, the development cost is low, the method is suitable for an industrialized amplification process route, and the nicotinamide product with high purity and low nicotinic acid content is prepared, so that the method has great scientific research significance and economic value.

Disclosure of Invention

The invention aims to solve the defects of the existing synthesis method, provides a recombinant nitrile hydratase and application thereof in preparing high-purity nicotinamide by coupling ion exchange resin, and aims to obtain low-cost and high-efficiency nitrile hydratase by modifying and optimizing nitrile hydratase-producing engineering bacteria, and simultaneously prepare high-purity nicotinamide by coupling ion exchange resin biological method, so that the cost of the nitrile hydratase is reduced, the enzyme activity and the thermal stability are improved, and meanwhile, a high-purity product is obtained.

The technical scheme adopted by the invention is as follows:

the invention provides a recombinant nitrile hydratase, and the amino acid sequence of the recombinant nitrile hydratase is shown as SEQ ID NO. 1.

The nucleotide sequence of the recombinant nitrile hydratase encoding gene is shown as SEQ ID NO. 4.

The invention obtains a nitrile hydratase gene cluster sequence Nitrile hydrataseH-Nhase (GenBank: D67027.1) from rhodococcus rhodochrous (Rhodococcusrhodochrous J1) according to NCBI search, comprises three parts of beta subunit (NHase-B), alpha subunit (NHase-A) and activator protein gene (NHase-G), and the beta subunit (NHase-B), the alpha subunit (NHase-A) and the activator protein gene (NHase-G) in the gene cluster are recombined after being optimized by escherichia coli codons to obtain the recombined nitrile hydratase.

The invention also relates to the recombinant hydratase encoding gene, a recombinant vector containing the nitrile hydratase encoding gene, and recombinant genetic engineering bacteria obtained by utilizing the recombinant vector transformation, wherein the engineering bacteria connect the synthetic gene to the vector pET-28a ⁺ And transformed into competent cells of escherichia coli BL21 (DE 3) to obtain recombinant escherichia coli genetically engineered bacteria.

The invention provides an application of recombinant nitrile hydratase coupled ion exchange resin in preparing nicotinamide, which comprises the following specific application methods: the concentrated solution of supernatant obtained by fermenting and culturing wet bacteria of recombinant nitrile hydratase gene engineering bacteria is taken as a catalyst, 3-cyanopyridine is taken as a substrate, ion exchange resin is added, phosphate buffer solution with pH value of 6-9 (preferably pH 7.0) is taken as a reaction medium to form a reaction system, hydration reaction is carried out at 20-30 ℃ and 100-1000rpm, and after the reaction is complete, the reaction solution is separated and purified to obtain the product nicotinamide. In the reaction system, the catalyst is used in an amount of 1-10g/L (preferably 2 g/L) based on the weight of wet bacterial cells before crushing, the final concentration of the substrate is 50-200mM (preferably 100 mM), and the addition amount of the ion exchange resin is 100-400g/L (preferably 100 g/L).

Further, the reaction time is preferably 5min to 180min, more preferably the reaction conditions are 20 to 25℃and 800 to 1000rpm for 30min.

Further, the buffer was Na at pH7.0, 0.2mM ₂ HPO ₄ /NaH ₂ PO ₄ Buffer solution.

Further, the ion exchange resin is an anion exchange resin (D101, D201, D301, D311, a764, JKA 915), preferably a D201 strongly basic anion exchange resin.

Further, the catalyst is prepared as follows: inoculating recombinant nitrile hydratase genetic engineering bacteria into liquid LB culture medium containing kanamycin (Kan) resistance with final concentration of 50 mug/mL, and culturing at 37 ℃ for 10h; the seed solution after culture was transferred to a new liquid LB medium containing kanamycin (Kan) resistance at a final concentration of 50. Mu.g/mL at an inoculum size of 1% by volume, and OD was cultured at 37 ℃ ₆₀₀ To a final concentration of 0.6-0.8, IPTG was added to a final concentration of 0.5mM, followed by 100mM CoCl ₂ Aqueous solution to CoCl ₂ Culturing at 28 deg.C for 12-16 hr at final concentration of 0.4-0.5mM, centrifuging at 8000-9000rpm at 4deg.C for 10min, collecting thallus, washing thallus with PBS buffer at pH7.0 for 2 times, centrifuging at 8000-9000rpm at 4deg.C for 10min, and collecting wet thallus; the wet thalli are subjected to ultrasonic disruption after being resuspended by PBS buffer solution, centrifugated (centrifugated for 10min at 12000rpm and 4 ℃), supernatant is taken, and concentrated to 10-30% (preferably 20%) of the volume of the disruption mixed solution, thus obtaining crude enzyme solution; the ultrasonic crushing condition is that the ultrasonic crushing is carried out for 5min at 300W, each ultrasonic is continued for 1s, and the interval is 2s; LB medium composition: 10g/L of tryptone, 5g/L of yeast powder and 10g/L of sodium chloride, wherein the solvent is deionized water, and the pH value is 7.

Because of the specificity of the nucleotide sequence, any variant of the polynucleotide shown in SEQ ID NO.4, as long as it has more than 70% homology with the polynucleotide and has the same function, falls within the scope of the present invention. A variant of the polynucleotide refers to a polynucleotide sequence having one or more nucleotide changes. Variants of the polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants, including substitution variants, deletion variants, and insertion variants. As known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the encoded amino acid.

In addition, it can be compared with SEQ ID NO:4 (preferably having at least 50% homology, preferably 70% homology), and also within the scope of the present invention, in particular polynucleotides which hybridize under stringent conditions to a nucleotide sequence according to the present invention. The term "stringent conditions" means: (1) Hybridization and elution at lower ionic strength and higher temperature, e.g., 0.2ssc,0.1% sds,60 ℃; or (2) adding denaturant such as 50% (v/v) formamide, 0.1% calf serum, 0.1% Ficoll, 42 ℃ during hybridization; or (3) hybridization only occurs when the homology between the two sequences is at least 95%, more preferably 97% or more. And, the protein encoded by the hybridizable polynucleotide hybridizes to the sequence of SEQ ID NO:1 has the same biological function and activity.

Compared with the prior art, the invention has the beneficial effects that: the invention provides a recombinant nitrile hydratase, wherein the encoding gene of the recombinant nitrile hydratase is combined by different subunits and then can be connected with an expression vector to construct an intracellular expression recombinant plasmid containing the gene, and then the intracellular expression recombinant plasmid is transformed into an escherichia coli strain to obtain recombinant escherichia coli. Compared with the wild strain, the expression intensity, the nitrile hydratase activity, the heat stability, the substrate and the product tolerance of the wild strain are obviously improved.

In the same way, the adsorption method of the recombinant nitrile hydratase catalytic coupling ion exchange resin of the invention adsorbs more than 95% of nicotinic acid impurities on the basis of not influencing the conversion rate, and obtains nicotinamide products with purity of more than 99.99%.

Drawings

FIG. 1 is a schematic representation of a nitrile hydratase catalyzed 3-cyanopyridine preparation of nicotinamide.

FIG. 2 is a diagram showing the prediction of the secondary structure of mRNA in the translation initiation region of nitrile hydratase.

FIG. 3 is a diagram showing construction of a nitrile hydratase recombinant plasmid constructed in example 1, a being pET28a-NHaseYpB ₁ B is pET28a-NHaseYpB ₂ C is pET28a-NHaseYpB ₃ D is pET28a-NHaseYpB ₄ 。

FIG. 4 is a liquid chromatogram of the adsorption reaction solution of the enzyme-catalyzed coupled ion exchange resin of example 4.

FIG. 5 is a graph showing the progress of the enzyme-catalyzed coupling resin reaction and the content of nicotinic acid as a by-product in example 4.

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto, and variations in the method according to these embodiments by those skilled in the art are included in the scope of the invention.

Example 1, nitrile hydratase Gene cloning and engineering bacterium construction

1. Engineering bacterium pET28a-NHaseYpB ₁

(1) Wild type nitrile hydratase

According to NCBI, nitrile hydratase (nitrile hydratase H-Nhase, genBank: D67027.1) derived from rhodococcus rhodochrous (R.rhodochrous J1) contains three parts of beta subunit (NHase-B), alpha subunit (NHase-A) and activator gene (NHase-G), and the three fragments are synthesized by Beijing-family biological science and technology Co., ltd after codon optimization according to E.coli preference, the nucleotide sequence of the beta subunit (NHase-B) is shown as 1-690 in SEQ ID NO.2, the nucleotide sequence of the alpha subunit (NHase-A) is shown as 691-1302 in SEQ ID NO.2, and the nucleotide sequence of the activator gene (NHase-G) is shown as 697 in SEQ ID NO. 2.

(2) Fragments

Beta subunit fragment: the beta subunit (NHase-B) fragment of the nitrile hydratase gene synthesized in step (1) is used as a template, and B in Table 1 is used _1-1 And B _1-2 As primers, PCR amplification was performed using the system shown in Table 2, and a PCR product, namely a beta subunit fragment, was obtained.

Alpha subunit fragment: the alpha subunit (NHase-A) fragment of the nitrile hydratase gene synthesized in step (1) is used as a template, and B in Table 1 is used _1-3 And B _1-4 As primers, PCR amplification was performed using the system shown in Table 2, and the obtained PCR product was an alpha subunit fragment.

Activator protein fragment: the nitrile hydratase gene activator protein (NHase-G) fragment synthesized in step (1) is used as a template, and B in Table 1 is used _1-5 And B _1-6 As primers, PCR amplification was performed using the system shown in Table 2, and the obtained PCR product was an activin fragment.

(3) The alpha subunit fragment, the beta subunit fragment and the activin fragment are subjected to multi-segment homologous recombination by adopting a vector pET-28a and adopting a 'Northenozan ClonExpress Ultra One Step Cloning Kit C' rapid cloning kit according to the system shown in the table 3, and the reaction is carried out for 15min at 50 ℃, wherein the nucleotide sequence is shown as SEQ ID NO.2 (wherein 1-690 nucleotide is beta subunit (NHase-B) coding gene, 691-1302 nucleotide is alpha subunit (NHase-A) coding gene and 1303-1617 nucleotideThe acid is the gene encoding activator protein (NHase-G) to obtain recombinant plasmid pET28a-NHaseYpB ₁ (a in fig. 3); cooling to 4 ℃ and preserving heat.

SEQ ID NO.2：

(4) Recombinant nitrile hydratase genetic engineering bacteria

Cloning the product pET28a-NHaseYpB from the step (3) in one step ₁ After E.coil BL21 (DE 3) competent cells are transformed, selecting a transformant, inoculating the transformant to 5mL of LB medium, culturing for 12 hours at 37 ℃, sequencing bacterial liquid by Beijing engine biotechnology Co., ltd, and obtaining a recombinant nitrile hydratase genetic engineering bacterium expressed from R.rhodochrous J1 with correct sequencing result, namely engineering bacterium E.coil BL21 (DE 3) -pET28a-NHaseYpB ₁ 。

PCR reaction conditions: (1) pre-denaturation at 98℃for 5min. (2) Denaturation at 98℃for 10s, annealing at 55℃for 15s, extension at 72℃for 20s, and cycling 30 times. (3) Extending at 72℃for 5min. (4) Preserving heat at 4 ℃.

TABLE 1 recombinant plasmid NHaseYpB ₁ Construction of primers

TABLE 2 PCR amplification System

Note that: the PCR was performed using a 20. Mu.L system, and the gene was cloned using a 50. Mu.L system.

TABLE 3 homologous recombination System

Multiple fragment homologous recombination: optimal cloning vector usage = [0.02 cloning vector base pair number ] ng, optimal usage per fragment [0.02 cloning vector base pair number ] ng.

2. Engineering bacterium E.coilBL21 (DE 3) -pET28a-NHaseYpB ₂

Engineering bacteria E.coil BL21 (DE 3) -pET28a-NHaseYpB ₁ The genome was used as a template, with B in Table 4 _2-1 And B _2-2 For the primer (the Ribosome Binding Site (RBS) was replaced with TAAGGAGGATATAG, the secondary structure of the mRNA in the translation initiation region after the replacement was shown in FIG. 2), PCR amplification was performed using the amplification system of Table 2 to obtain a PCR product, namely, a beta subunit fragment.

Engineering bacteria E.coil BL21 (DE 3) -pET28a-NHaseYpB ₁ The genome was used as a template, with B in Table 4 _2-3 And B _2-4 As primers (RBS was replaced with TAAGGAGGATATAG), PCR amplification was performed using the system shown in Table 2 to obtain PCR products, i.e., alpha subunit fragments.

Engineering bacteria E.coil BL21 (DE 3) -pET28a-NHaseYpB ₁ The genome was used as a template, with B in Table 4 _2-5 And B _2-6 As primers (RBS was replaced with TAAGGAGGATATAG), PCR amplification was performed using the system shown in Table 2 to obtain PCR products, i.e., activator protein fragments.

The vector pET-28a is adopted, alpha subunit fragment, beta subunit fragment and activator protein fragment are subjected to multi-segment homologous recombination by adopting a 'Noruzan ClonExpress Ultra One Step Cloning Kit C' rapid cloning kit according to the system shown in the table 3, and the reaction is carried out for 15min at 50 ℃ to obtain recombinant plasmid pET28a-NHaseYpB ₂ (b in fig. 3); cooling to 4 ℃ and preserving heat.

The one-step cloning product pET28a-NHaseYpB ₂ After E.coil BL21 (DE 3) competent cells are transformed, selecting a transformant, inoculating the transformant to 5mL of LB medium, culturing for 12 hours at 37 ℃, sequencing bacterial liquid by Beijing engine biotechnology Co., ltd, and obtaining a recombinant nitrile hydratase genetic engineering bacterium expressed from R.rhodochrous J1 with correct sequencing result, namely engineering bacterium E.coil BL21 (DE 3) -pET28a-NHaseYpB ₂ The nucleotide sequence is shown as SEQ ID NO.3, and the 1-14, 705-718, 1331-1344 nucleotides in the nucleotide sequence shown as SEQ ID NO.3 are all RBS,15-704 nucleotides are beta subunit (NHase-B), 719-1330 nucleotides are alpha subunit (NHase-A), 1345-1659 nucleotides are activator protein gene (NHase-G).

SEQ ID NO.3：

3. Engineering bacterium E.coilBL21 (DE 3) -pET28a-NHaseYpB ₃

Engineering bacteria E.coil BL21 (DE 3) -pET28a-NHaseYpB ₂ The genome was used as a template, with B in Table 5 _3-1 And B _3-2 For the primer (inserting fusion label SKIK: TCTAAAATAAAA), PCR amplification is carried out by adopting the amplification system of the table 2, and the PCR product is obtained, namely the beta subunit fragment.

Engineering bacteria E.coil BL21 (DE 3) -pET28a-NHaseYpB ₂ The genome was used as a template, with B in Table 5 _3-3 And B _3-4 For primer (inserting fusion label SKIK: TCTAAAATAAAA), PCR amplification is carried out by adopting a system shown in Table 2 to obtain PCR product, namely alpha subunit fragment.

Engineering bacteria E.coil BL21 (DE 3) -pET28a-NHaseYpB ₂ The genome was used as a template, with B in Table 5 _3-5 And B _3-6 For the primer (inserted fusion tag SKIK: TCTAAAATAAAA), PCR amplification was performed using the system shown in Table 2 to obtain PCR product, i.e., activator protein fragment.

The vector pET-28a is adopted, alpha subunit fragment, beta subunit fragment and activator protein fragment are subjected to multi-segment homologous recombination by adopting a 'Noruzan ClonExpress Ultra One Step Cloning Kit C' rapid cloning kit according to the system shown in the table 3, and the reaction is carried out for 15min at 50 ℃ to obtain recombinant plasmid pET28a-NHaseYpB ₃ (c in FIG. 3); cooling to 4 ℃ and preserving heat.

The one-step cloning product pET28a-NHaseYpB ₃ After E.coil BL21 (DE 3) competent cells are transformed, selecting a transformant, inoculating the transformant to 5mL of LB culture medium, culturing for 12 hours at 37 ℃, sequencing bacterial liquid by Beijing engine biotechnology limited company, and obtaining recombinant nitrile hydratase genetic engineering bacteria expressing R.rhodochrous J1 by correct sequencing result, namely engineering bacteria E.coil BL21(DE3)-pET28a-NHaseYpB ₃ The nucleotide sequence is shown as SEQ ID NO.4, wherein nucleotides 1-14, 705-718 and 1343-1356 are RBS, nucleotides 15-704 are beta subunit (NHase-B), nucleotides 719-1342 are alpha subunit (NHase-A), nucleotides 1356-1683 are activator protein gene (NHase-G), and nucleotides 722-733 and 1360-1371 are fusion tags. The amino acid sequence is shown as SEQ ID NO.1 (wherein amino acids 1-229 are beta subunit (NHase-B), amino acids 230-436 are alpha subunit (NHase-A), and amino acids 437-544 are activator protein (NHase-G)).

SEQ ID NO.4：

SEQ ID NO.1：

MDGIHDTGGMTGYGPVPYQKDEPFFHYEWEGRTLSILTWMHLKGISWWDKSRFFRE SMGNENYVNEIRNSYYTHWLSAAERILVADKIITEEERKHRVQEILEGRYTDRKPSRKFDPAQIEKAIERLHEPHSLALPGAEPSFSLGDKIKVKSMNPLGHTRCPKYVRNKIGEIVAYHGC QIYPESSSAGLGDDPRPLYTVAFSAQELWGDDGNGKDVVCVDLWEPYLISAMSKIKSEHVNKYTEYEARTKAIETLLYERGLITPAAVDRVVSYYENEIGPMGGAKVVAKSWVDPEYRK WLEEDATAAMASLGYAGEQAHQISAVFNDSQTHHVVVCTLCSCYPWPVLGLPPAWYKSMEYRSRVVADPRGVLKRDFGFDIPDEVEVRVWDSSSEIRYIVIPERPAGTDGWSEEELTKL VSRDSMIGVSNALTPQEVIVMSKIKSEDTLTDRLPATGTAAPPRDNGELVFTEPWEATAFGVAIALSDQKSYEWEFFRQRLIHSIAEANGCEAYYESWTKALEASVVDSGLISEDEIRERMES MAIID。

4. Engineering bacterium E.coilBL21 (DE 3) -pET28a-NHaseYpB ₄

Engineering bacteria E.coil BL21 (DE 3) -pET28a-NHaseYpB ₃ The genome was used as a template, with B in Table 6 _4-1 And B _4-2 PCR amplification is carried out by adopting an amplification system of the table 2 as a primer (flexible short peptide chain Linker: GGSGGGSGGGSG), and a PCR product is obtained, namely the beta subunit fragment.

Engineering bacteria E.coil BL21 (DE 3) -pET28a-NHaseYpB ₃ The genome was used as a template, with B in Table 6 _4-3 And B _4-4 As a primer (flexible short peptide chain Linker: GGSGGGSGGGSG), PCR amplification was carried out using the system shown in Table 2 to obtain a PCR product, namely, an alpha subunit fragment.

Engineering bacteria E.coil BL21 (DE 3) -pET28a-NHaseYpB ₃ The genome was used as a template, with B in Table 6 _4-5 And B _4-6 As primers, PCR amplification was performed using the system shown in Table 2 to obtain PCR products, i.e., activator protein fragments.

The vector pET-28a is adopted, alpha subunit fragment, beta subunit fragment and activator protein fragment are subjected to multi-segment homologous recombination by adopting a 'Noruzan ClonExpress Ultra One Step Cloning Kit C' rapid cloning kit according to the system shown in the table 3, and the reaction is carried out for 15min at 50 ℃ to obtain recombinant plasmid pET28a-NHaseYpB ₄ (d in FIG. 3); cooling to 4 ℃ and preserving heat.

The one-step cloning product pET28a-NHaseYpB ₄ After E.coil BL21 (DE 3) competent cells are transformed, selecting a transformant, inoculating the transformant to 5mL of LB medium, culturing for 12 hours at 37 ℃, sequencing bacterial liquid by Beijing engine biotechnology Co., ltd, and obtaining a recombinant nitrile hydratase genetic engineering bacterium expressed from R.rhodochrous J1 with correct sequencing result, namely engineering bacterium E.coil BL21 (DE 3) -pET28a-NHaseYpB ₄ The nucleotide sequence is shown as SEQ ID NO.5, wherein nucleotides 1-14 and 1359-1372 are RBS, nucleotides 15-701 are beta subunit (NHase-B), nucleotides 750-1358 are alpha subunit (NHase-A), nucleotides 1373-1699 are activator protein gene (NHase-G), nucleotides 738-749 and 1376-1387 are fusion tag SKIK, and nucleotides 702-737 are Linker.

SEQ ID NO.5：

TABLE 4 recombinant plasmid NHaseYpB ₂ Construction of primers

TABLE 5 recombinant plasmid NHaseYpB ₃ Construction of primers

TABLE 6 recombinant plasmid NHaseYpB ₄ Construction of primers

Example 2 inducible expression of recombinant nitrile hydratase Gene engineering bacteria

Recombinant E.coli engineering bacteria E.coil BL21 (DE 3) -pET28a-NHaseYpB with correct sequencing of example 1 ₁ 、E.coil BL21(DE3)-pET28a-NHaseYpB ₂ 、E.coil BL21(DE3)-pET28a-NHaseYpB ₃ 、E.coil BL21(DE3)-pET28a-NHaseYpB ₄ Each of the cells was inoculated into 10mL of a liquid LB medium containing Kan (final concentration: 50. Mu.g/mL), and cultured at 37℃for 10 hours. Transferring the cultured seed solution into new 100mL liquid LB medium containing Kan (final concentration of 50 μg/mL) at 1% inoculation amount, culturing under the same condition until the bacterial liquid OD600 is 0.6, adding IPTG with final concentration of 0.5mM, and adding 400 μl of 100mM CoCl ₂ Aqueous solution (final concentration 0.4 mM), induced at 28℃for 12h; the cells were collected by centrifugation at 9000rpm for 10 min. The cells were washed 2 times with PBS buffer at pH7, and centrifuged at 9000rpm for 10min to collect wet cells. 0.2g of wet thalli is taken and resuspended by using 10mL of PBS buffer solution with pH7, the resuspension bacterial solution is placed in a 10mL centrifuge tube, the centrifuge tube is placed on ice, the ultrasonic disruption is carried out for 5min by 300W, each ultrasonic disruption lasts for 1s, and the interval is 2s, thus obtaining the cell disruption suspension. 1mL of the cell disruption suspension was centrifuged at 12000rpm at 4℃for 10min, and the supernatant was transferred to a new centrifuge tube and concentrated to obtain 200. Mu.L of crude enzyme solution. The specific enzyme activities were measured, and the results are shown in Table 7, in which the recombinant E.coli engineering bacteria E.coil BL21 (DE 3) -pET28a-NHaseYpB ₃ The activity is highest.

Enzyme activity detection method: enzyme activity detection of recombinant nitrile hydratase engineering bacteria by using 3-cyanopyridine as model substrate. The enzyme activity detection reaction system is 1mL:100mM Na ₂ HPO ₄ As a reaction medium, a citric acid buffer (pH 7.0) was used, 3-cyanopyridine was used as a substrate at a final concentration of 100mM, 20. Mu.L of a crude enzyme solution (corresponding to 0.002g of wet cells before disruption) was added thereto, and the reaction was terminated by stirring at 25℃for 5 minutes at 700rpm, and 200. Mu.L of the reaction solution was added to 1mL of methanol. Detecting the nicotinamide peak area of the product, the nicotinic acid peak area of the byproduct and the 3-cyanopyridine peak area of the substrate by high performance liquid chromatography, and obtaining the nicotinamide content in the product according to standard curves (nicotinamide: y=823756x+6312, 3-cyanopyridine: y=8714288x+4062, nicotinic acid: y=13882 x-361).

The liquid phase detection method comprises the following steps: chromatographic column: welchromC18 column (250X 4.6 mm) Zhejiang Yuan Xue materials science and technology Co., ltd., mobile phase: methanol-disodium hydrogen phosphate solution (20 mmol/L) =8:92 (v/v); wavelength: 268nm; flow rate: 1.0mL/min, UV-visible detector Waters 2487, waters, inc.

Definition of enzyme activity unit (U): under the reaction conditions, the amount of enzyme required to catalyze 1. Mu. Mol of nicotinamide per minute for 3-cyanopyridine is one enzyme activity unit, denoted by U. Specific enzyme activity definition: the crude enzyme protein has an enzyme activity expressed as U/mL.

TABLE 7 specific enzyme Activity of recombinant bacteria

Example 3 influence of resin on enzymatic reactions

Pretreatment of resin: (1) Resins D201, D301, JKA915, BA765, D101, and D311 (all purchased from Jiangsu Kai resin chemical Co., ltd.) in Table 8 were immersed in a 10% aqueous NaCl solution having a mass concentration of about 2 times the resin volume in a beaker, the resin layer was stirred at a low rotation speed of 20rpm for 1 hour, the liquid surface was placed on the resin layer at 200-300mm for immersion for 20 hours, and then rinsed with clear water until the water was colorless. (2) Then back washing is carried out to remove tiny impurities mixed in the resin, after the resin layer is soaked for 1h by using 4% HCl aqueous solution which is approximately equal to 2 times of the volume of the resin, the liquid level is placed on the resin layer at 200-300mm for soaking for 2-4 h, and the resin layer is washed by clean water until the pH value of the effluent is 5-6. (3) Soaking the resin layer with 2% NaOH water solution with mass concentration of about 2 times of the volume of the resin for 1h, placing the liquid surface at 200-300mm above the resin layer, soaking for 2-4 h, and washing with deionized water until the pH value of the effluent is 7-9.

Enzyme-coupled resin preparation of nicotinamide: mu.L (corresponding to 0.002g of wet cell before disruption) of recombinant E.coil BL21 (DE 3) -pET28a-NHaseYpB obtained in example 2 was taken ₃ Into a 2mL EP tube, 880. Mu.L of PB buffer (pH 7.0,0.2 mM) was added as a reaction solvent, then 100. Mu.L of 3-cyanopyridine (100 mM final concentration) was added, and 0.2g of pretreated resins D201, D301, JKA915, BA765, D101 and D311 (all obtained from Jiangsu Kai resin chemical Co., ltd.) were added to form a 1mL reaction system, and the reaction system was placed in a constant temperature mixer at 20℃and 1000rpm to react for 30 minutes. The blank was prepared without adding resin. After the completion of the reaction, the reaction mixture was quenched with 1mL of methanol. 10. Mu.L of the reaction solution was dissolved in 1mL of a mobile phase, and the residual amount of 3-cyanopyridine, the production amount of nicotinamide and the production amount of nicotinic acid as a by-product were measured by using the HPLC method described in example 2, whereby the enzyme-catalyzed hydrolysis activity and the purity of the product were estimated, and the experimental results are shown in Table 8. The D201 resin has the least influence on a reaction system, can selectively adsorb the by-product nicotinic acid, has the nicotinamide purity of 99.99 percent under a coupling system, has the by-product nicotinic acid content of 0.004 percent, and has the best reaction result.

TABLE 8 results of nitrile hydratase catalytic reactions under different resin adsorption conditions

Example 4 preparation of high purity nicotinamide by recombinant nitrile hydratase catalytic coupled ion exchange resin adsorption in a thermostatic waterbath reaction kettle, the enzyme catalytic reaction system was expanded to 1L: the reaction solvent was Na at pH7.0, 0.2mM ₂ HPO ₄ /NaH ₂ PO ₄ The buffer solution was added with 3-cyanopyridine as a substrate at a final concentration of 100mM, and 20mL (corresponding to 2g of wet cells before disruption) of the recombinant large intestine obtained in the method of example 2 was addedBacillus E.coil BL21 (DE 3) -pET28a-NHaseYpB ₃ And adding 100g of D201 strong-base anion exchange resin pretreated by the method of example 3 into the crude enzyme solution, and reacting for 30min in a constant-temperature water bath reaction kettle at 25 ℃ with stirring speed of 150 rpm. The reaction solution was taken every 5min and tested by the high performance liquid chromatography method described in example 2, and the substrate conversion and the content of by-product nicotinic acid were calculated, and the results are shown in FIG. 4. The reaction sequence diagram of the recombinant nitrile hydratase catalytic coupling ion exchange resin adsorption to prepare high purity nicotinamide is shown in figure 5. The conversion rate reaches more than 99.99 percent under a coupling system, and the content of the impurity nicotinic acid is 0.004 percent.

Sequence listing

<110> Zhejiang university of industry

<120> recombinant nitrile hydratase and use thereof in preparation of nicotinamide by coupling ion exchange resin

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 544

<212> PRT

<213> rhodococcus rhodochrous (rhodococcus rhodochrous)

<400> 1

Met Asp Gly Ile His Asp Thr Gly Gly Met Thr Gly Tyr Gly Pro Val

1 5 10 15

Pro Tyr Gln Lys Asp Glu Pro Phe Phe His Tyr Glu Trp Glu Gly Arg

20 25 30

Thr Leu Ser Ile Leu Thr Trp Met His Leu Lys Gly Ile Ser Trp Trp

35 40 45

Asp Lys Ser Arg Phe Phe Arg Glu Ser Met Gly Asn Glu Asn Tyr Val

50 55 60

Asn Glu Ile Arg Asn Ser Tyr Tyr Thr His Trp Leu Ser Ala Ala Glu

65 70 75 80

Arg Ile Leu Val Ala Asp Lys Ile Ile Thr Glu Glu Glu Arg Lys His

85 90 95

Arg Val Gln Glu Ile Leu Glu Gly Arg Tyr Thr Asp Arg Lys Pro Ser

100 105 110

Arg Lys Phe Asp Pro Ala Gln Ile Glu Lys Ala Ile Glu Arg Leu His

115 120 125

Glu Pro His Ser Leu Ala Leu Pro Gly Ala Glu Pro Ser Phe Ser Leu

130 135 140

Gly Asp Lys Ile Lys Val Lys Ser Met Asn Pro Leu Gly His Thr Arg

145 150 155 160

Cys Pro Lys Tyr Val Arg Asn Lys Ile Gly Glu Ile Val Ala Tyr His

165 170 175

Gly Cys Gln Ile Tyr Pro Glu Ser Ser Ser Ala Gly Leu Gly Asp Asp

180 185 190

Pro Arg Pro Leu Tyr Thr Val Ala Phe Ser Ala Gln Glu Leu Trp Gly

195 200 205

Asp Asp Gly Asn Gly Lys Asp Val Val Cys Val Asp Leu Trp Glu Pro

210 215 220

Tyr Leu Ile Ser Ala Met Ser Lys Ile Lys Ser Glu His Val Asn Lys

225 230 235 240

Tyr Thr Glu Tyr Glu Ala Arg Thr Lys Ala Ile Glu Thr Leu Leu Tyr

245 250 255

Glu Arg Gly Leu Ile Thr Pro Ala Ala Val Asp Arg Val Val Ser Tyr

260 265 270

Tyr Glu Asn Glu Ile Gly Pro Met Gly Gly Ala Lys Val Val Ala Lys

275 280 285

Ser Trp Val Asp Pro Glu Tyr Arg Lys Trp Leu Glu Glu Asp Ala Thr

290 295 300

Ala Ala Met Ala Ser Leu Gly Tyr Ala Gly Glu Gln Ala His Gln Ile

305 310 315 320

Ser Ala Val Phe Asn Asp Ser Gln Thr His His Val Val Val Cys Thr

325 330 335

Leu Cys Ser Cys Tyr Pro Trp Pro Val Leu Gly Leu Pro Pro Ala Trp

340 345 350

Tyr Lys Ser Met Glu Tyr Arg Ser Arg Val Val Ala Asp Pro Arg Gly

355 360 365

Val Leu Lys Arg Asp Phe Gly Phe Asp Ile Pro Asp Glu Val Glu Val

370 375 380

Arg Val Trp Asp Ser Ser Ser Glu Ile Arg Tyr Ile Val Ile Pro Glu

385 390 395 400

Arg Pro Ala Gly Thr Asp Gly Trp Ser Glu Glu Glu Leu Thr Lys Leu

405 410 415

Val Ser Arg Asp Ser Met Ile Gly Val Ser Asn Ala Leu Thr Pro Gln

420 425 430

Glu Val Ile Val Met Ser Lys Ile Lys Ser Glu Asp Thr Leu Thr Asp

435 440 445

Arg Leu Pro Ala Thr Gly Thr Ala Ala Pro Pro Arg Asp Asn Gly Glu

450 455 460

Leu Val Phe Thr Glu Pro Trp Glu Ala Thr Ala Phe Gly Val Ala Ile

465 470 475 480

Ala Leu Ser Asp Gln Lys Ser Tyr Glu Trp Glu Phe Phe Arg Gln Arg

485 490 495

Leu Ile His Ser Ile Ala Glu Ala Asn Gly Cys Glu Ala Tyr Tyr Glu

500 505 510

Ser Trp Thr Lys Ala Leu Glu Ala Ser Val Val Asp Ser Gly Leu Ile

515 520 525

Ser Glu Asp Glu Ile Arg Glu Arg Met Glu Ser Met Ala Ile Ile Asp

530 535 540

<210> 2

<211> 1617

<212> DNA

<213> rhodococcus rhodochrous (rhodococcus rhodochrous)

<400> 2

atggatggta ttcatgatac aggtggtatg accggttatg gtccggtgcc gtatcagaaa 60

gatgaaccgt tttttcatta tgaatgggaa ggtcgtacgc tgagcattct gacctggatg 120

catctgaaag gtattagctg gtgggataaa agtcgttttt ttcgtgaaag catgggtaat 180

gaaaattatg ttaacgaaat ccgtaacagt tattataccc attggctgag cgcggcggaa 240

cgtatcctgg ttgcagataa aattattacc gaagaagaac gcaaacatcg tgtgcaggaa 300

atcctggaag gtcgttatac agatcgcaaa ccgagccgta aatttgatcc tgcacagatt 360

gaaaaagcaa ttgaacgcct gcatgaaccg catagcctgg cactgcctgg tgcagaacca 420

tcttttagtc tgggtgataa aattaaagtg aaaagcatga acccactggg tcatacccgt 480

tgtccgaaat atgttcgtaa caaaattggt gaaatcgttg catatcatgg ttgtcagatc 540

tatccggaaa gtagcagcgc aggtctgggt gatgatccac gcccgctgta taccgttgcc 600

tttagcgccc aggaactgtg gggtgatgat ggtaatggta aagatgtggt ttgtgttgat 660

ctgtgggaac cgtatctgat tagcgcataa atgagcgaac acgtgaacaa atataccgaa 720

tatgaagcac gcaccaaagc cattgaaacc ctgctgtacg aacgcggcct gatcaccccg 780

gcagcggttg accgcgttgt gagctactac gaaaatgaaa ttggcccgat gggcggagca 840

aaagttgttg caaaaagctg ggtggatccg gaatatcgta aatggctgga agaagatgca 900

accgcagcaa tggcaagcct ggggtatgca ggggaacagg cgcatcagat tagcgcagtg 960

tttaatgata gccagacgca tcatgttgtt gtttgtaccc tgtgtagttg ttatccgtgg 1020

ccggttctgg gtctgcctcc ggcatggtat aaaagcatgg aatatcgcag ccgtgttgtt 1080

gccgatccac gtggtgttct gaaacgtgat tttggctttg atattccgga cgaggttgaa 1140

gttcgtgttt gggatagtag tagcgaaatt cgttatattg ttatcccgga acgtccggca 1200

gggaccgatg ggtggagcga ggaagaactg acaaaactgg ttagccgtga tagtatgatt 1260

ggtgtgagca atgcactgac accgcaggaa gtgatcgttt aaatgagcga agacacgctg 1320

accgaccgcc tgccggcaac cggaaccgca gcaccaccgc gtgacaacgg tgaactggtt 1380

tttaccgaac cgtgggaagc aaccgcattt ggcgttgcaa ttgcactgag cgaccaaaaa 1440

tcatatgaat gggaattttt ccgccaacgt ctgatccata gcattgcgga ggcaaatggt 1500

tgcgaggcat attatgaaag ctggacgaaa gcactggagg caagcgttgt tgatagcggt 1560

ctgattagcg aagatgaaat tcgtgaacgt atggaaagca tggcaattat tgattaa 1617

<210> 3

<211> 1659

<212> DNA

<213> rhodococcus rhodochrous (rhodococcus rhodochrous)

<400> 3

taaggaggat atagatggat ggtattcatg atacaggtgg tatgaccggt tatggtccgg 60

tgccgtatca gaaagatgaa ccgttttttc attatgaatg ggaaggtcgt acgctgagca 120

ttctgacctg gatgcatctg aaaggtatta gctggtggga taaaagtcgt ttttttcgtg 180

aaagcatggg taatgaaaat tatgttaacg aaatccgtaa cagttattat acccattggc 240

tgagcgcggc ggaacgtatc ctggttgcag ataaaattat taccgaagaa gaacgcaaac 300

atcgtgtgca ggaaatcctg gaaggtcgtt atacagatcg caaaccgagc cgtaaatttg 360

atcctgcaca gattgaaaaa gcaattgaac gcctgcatga accgcatagc ctggcactgc 420

ctggtgcaga accatctttt agtctgggtg ataaaattaa agtgaaaagc atgaacccac 480

tgggtcatac ccgttgtccg aaatatgttc gtaacaaaat tggtgaaatc gttgcatatc 540

atggttgtca gatctatccg gaaagtagca gcgcaggtct gggtgatgat ccacgcccgc 600

tgtataccgt tgcctttagc gcccaggaac tgtggggtga tgatggtaat ggtaaagatg 660

tggtttgtgt tgatctgtgg gaaccgtatc tgattagcgc ataataagga ggatatagat 720

gagcgaacac gtgaacaaat ataccgaata tgaagcacgc accaaagcca ttgaaaccct 780

gctgtacgaa cgcggcctga tcaccccggc agcggttgac cgcgttgtga gctactacga 840

aaatgaaatt ggcccgatgg gcggagcaaa agttgttgca aaaagctggg tggatccgga 900

atatcgtaaa tggctggaag aagatgcaac cgcagcaatg gcaagcctgg ggtatgcagg 960

ggaacaggcg catcagatta gcgcagtgtt taatgatagc cagacgcatc atgttgttgt 1020

ttgtaccctg tgtagttgtt atccgtggcc ggttctgggt ctgcctccgg catggtataa 1080

aagcatggaa tatcgcagcc gtgttgttgc cgatccacgt ggtgttctga aacgtgattt 1140

tggctttgat attccggacg aggttgaagt tcgtgtttgg gatagtagta gcgaaattcg 1200

ttatattgtt atcccggaac gtccggcagg gaccgatggg tggagcgagg aagaactgac 1260

aaaactggtt agccgtgata gtatgattgg tgtgagcaat gcactgacac cgcaggaagt 1320

gatcgtttaa taaggaggat atagatgagc gaagacacgc tgaccgaccg cctgccggca 1380

accggaaccg cagcaccacc gcgtgacaac ggtgaactgg tttttaccga accgtgggaa 1440

gcaaccgcat ttggcgttgc aattgcactg agcgaccaaa aatcatatga atgggaattt 1500

ttccgccaac gtctgatcca tagcattgcg gaggcaaatg gttgcgaggc atattatgaa 1560

agctggacga aagcactgga ggcaagcgtt gttgatagcg gtctgattag cgaagatgaa 1620

attcgtgaac gtatggaaag catggcaatt attgattaa 1659

<210> 4

<211> 1683

<212> DNA

<213> rhodococcus rhodochrous (rhodococcus rhodochrous)

<400> 4

taaggaggat atagatggat ggtattcatg atacaggtgg tatgaccggt tatggtccgg 60

tgccgtatca gaaagatgaa ccgttttttc attatgaatg ggaaggtcgt acgctgagca 120

ttctgacctg gatgcatctg aaaggtatta gctggtggga taaaagtcgt ttttttcgtg 180

aaagcatggg taatgaaaat tatgttaacg aaatccgtaa cagttattat acccattggc 240

tgagcgcggc ggaacgtatc ctggttgcag ataaaattat taccgaagaa gaacgcaaac 300

atcgtgtgca ggaaatcctg gaaggtcgtt atacagatcg caaaccgagc cgtaaatttg 360

atcctgcaca gattgaaaaa gcaattgaac gcctgcatga accgcatagc ctggcactgc 420

ctggtgcaga accatctttt agtctgggtg ataaaattaa agtgaaaagc atgaacccac 480

tgggtcatac ccgttgtccg aaatatgttc gtaacaaaat tggtgaaatc gttgcatatc 540

atggttgtca gatctatccg gaaagtagca gcgcaggtct gggtgatgat ccacgcccgc 600

tgtataccgt tgcctttagc gcccaggaac tgtggggtga tgatggtaat ggtaaagatg 660

tggtttgtgt tgatctgtgg gaaccgtatc tgattagcgc ataataagga ggatatagat 720

gtctaaaata aaaagcgaac acgtgaacaa atataccgaa tatgaagcac gcaccaaagc 780

cattgaaacc ctgctgtacg aacgcggcct gatcaccccg gcagcggttg accgcgttgt 840

gagctactac gaaaatgaaa ttggcccgat gggcggagca aaagttgttg caaaaagctg 900

ggtggatccg gaatatcgta aatggctgga agaagatgca accgcagcaa tggcaagcct 960

ggggtatgca ggggaacagg cgcatcagat tagcgcagtg tttaatgata gccagacgca 1020

tcatgttgtt gtttgtaccc tgtgtagttg ttatccgtgg ccggttctgg gtctgcctcc 1080

ggcatggtat aaaagcatgg aatatcgcag ccgtgttgtt gccgatccac gtggtgttct 1140

gaaacgtgat tttggctttg atattccgga cgaggttgaa gttcgtgttt gggatagtag 1200

tagcgaaatt cgttatattg ttatcccgga acgtccggca gggaccgatg ggtggagcga 1260

ggaagaactg acaaaactgg ttagccgtga tagtatgatt ggtgtgagca atgcactgac 1320

accgcaggaa gtgatcgttt aataaggagg atatagatgt ctaaaataaa aagcgaagac 1380

acgctgaccg accgcctgcc ggcaaccgga accgcagcac caccgcgtga caacggtgaa 1440

ctggttttta ccgaaccgtg ggaagcaacc gcatttggcg ttgcaattgc actgagcgac 1500

caaaaatcat atgaatggga atttttccgc caacgtctga tccatagcat tgcggaggca 1560

aatggttgcg aggcatatta tgaaagctgg acgaaagcac tggaggcaag cgttgttgat 1620

agcggtctga ttagcgaaga tgaaattcgt gaacgtatgg aaagcatggc aattattgat 1680

taa 1683

<210> 5

<211> 1699

<212> DNA

<213> rhodococcus rhodochrous (rhodococcus rhodochrous)

<400> 5

taaggaggat atagatggat ggtattcatg atacaggtgg tatgaccggt tatggtccgg 60

tgccgtatca gaaagatgaa ccgttttttc attatgaatg ggaaggtcgt acgctgagca 120

ttctgacctg gatgcatctg aaaggtatta gctggtggga taaaagtcgt ttttttcgtg 180

aaagcatggg taatgaaaat tatgttaacg aaatccgtaa cagttattat acccattggc 240

tgagcgcggc ggaacgtatc ctggttgcag ataaaattat taccgaagaa gaacgcaaac 300

atcgtgtgca ggaaatcctg gaaggtcgtt atacagatcg caaaccgagc cgtaaatttg 360

atcctgcaca gattgaaaaa gcaattgaac gcctgcatga accgcatagc ctggcactgc 420

ctggtgcaga accatctttt agtctgggtg ataaaattaa agtgaaaagc atgaacccac 480

tgggtcatac ccgttgtccg aaatatgttc gtaacaaaat tggtgaaatc gttgcatatc 540

atggttgtca gatctatccg gaaagtagca gcgcaggtct gggtgatgat ccacgcccgc 600

tgtataccgt tgcctttagc gcccaggaac tgtggggtga tgatggtaat ggtaaagatg 660

tggtttgtgt tgatctgtgg gaaccgtatc tgattagcgc aggtggtagc ggtggtggta 720

gcggtggtgg tagcggttct aaaataaaaa gcgaacacgt gaacaaatat accgaatatg 780

aagcacgcac caaagccatt gaaaccctgc tgtacgaacg cggcctgatc accccggcag 840

cggttgaccg cgttgtgagc tactacgaaa atgaaattgg cccgatgggc ggagcaaaag 900

ttgttgcaaa aagctgggtg gatccggaat atcgtaaatg gctggaagaa gatgcaaccg 960

cagcaatggc aagcctgggg tatgcagggg aacaggcgca tcagattagc gcagtgttta 1020

atgatagcca gacgcatcat gttgttgttt gtaccctgtg tagttgttat ccgtggccgg 1080

ttctgggtct gcctccggca tggtataaaa gcatggaata tcgcagccgt gttgttgccg 1140

atccacgtgg tgttctgaaa cgtgattttg gctttgatat tccggacgag gttgaagttc 1200

gtgtttggga tagtagtagc gaaattcgtt atattgttat cccggaacgt ccggcaggga 1260

ccgatgggtg gagcgaggaa gaactgacaa aactggttag ccgtgatagt atgattggtg 1320

tgagcaatgc actgacaccg caggaagtga tcgtttaata aggaggatat agatgtctaa 1380

aataaaaagc gaagacacgc tgaccgaccg cctgccggca accggaaccg cagcaccacc 1440

gcgtgacaac ggtgaactgg tttttaccga accgtgggaa gcaaccgcat ttggcgttgc 1500

aattgcactg agcgaccaaa aatcatatga atgggaattt ttccgccaac gtctgatcca 1560

tagcattgcg gaggcaaatg gttgcgaggc atattatgaa agctggacga aagcactgga 1620

ggcaagcgtt gttgatagcg gtctgattag cgaagatgaa attcgtgaac gtatggaaag 1680

catggcaatt attgattaa 1699

Claims

1. The recombinant nitrile hydratase is characterized in that the nucleotide sequence of the encoding gene of the recombinant nitrile hydratase is shown as SEQ ID NO. 4.

2. The recombinant nitrile hydratase encoding gene is characterized in that the nucleotide sequence of the recombinant nitrile hydratase encoding gene is shown as SEQ ID NO. 4.

3. A recombinant vector constructed from the coding gene of claim 2.

4. A recombinant genetically engineered bacterium prepared by transforming a host bacterium with the recombinant vector of claim 3.

5. Use of the recombinant nitrile hydratase of claim 1 for the preparation of nicotinamide by coupling ion exchange resins, wherein the method of use comprises: taking concentrated solution of supernatant obtained by fermenting and culturing wet bacteria obtained by fermenting and culturing recombinant nitrile hydratase genetic engineering bacteria as a catalyst, taking 3-cyanopyridine as a substrate, adding ion exchange resin, taking phosphoric acid buffer solution with pH value of 6-9 as a reaction medium to form a reaction system, carrying out hydration reaction at 20-30 ℃ and 100-1000rpm, and separating and purifying the reaction solution after the reaction is complete to obtain nicotinamide; the ion exchange resin is D201, D301 and JKA915.

6. The use according to claim 5, wherein the catalyst is used in an amount of 1 to 10g/L based on the weight of wet cells before disruption, the final concentration of the substrate is 50 to 200mM, and the amount of the ion exchange resin added is 100 to 400g/L.

7. The use according to claim 5, wherein the buffer solution is Na at pH7.0, 0.2mM ₂ HPO ₄ /NaH ₂ PO ₄ Buffer solution.

8. The use according to claim 5, wherein the catalyst is prepared by the following method: inoculating recombinant nitrile hydratase genetic engineering bacteria into a liquid LB culture medium containing kanamycin resistance with a final concentration of 50 mug/mL, and culturing for 10 hours at 37 ℃; the seed solution after culture was transferred to a new liquid LB medium containing 50. Mu.g/mL kanamycin resistance at a final concentration of 1% by volume of the inoculum size, and OD was cultured at 37 ℃ ₆₀₀ IPTG was added to a final concentration of 0.5mM at 0.6-0.8, followed by 100mM CoCl ₂ Aqueous solution to CoCl ₂ Culturing at 28 deg.C at final concentration of 0.4-0.5mM for 12-16h, centrifuging, washing with PBS buffer solution of pH7.0, re-suspending, ultrasonic crushing, centrifuging, collecting supernatant, and concentrating to 10-30% of the volume of the crushed mixture to obtain crude enzyme solution; ultrasonic crushing condition of 300WCrushing by sound for 5min, wherein each time of ultrasonic treatment lasts for 1s, and the interval is 2s; LB medium composition: 10g/L of tryptone, 5g/L of yeast powder, 10g/L of sodium chloride and g/L of deionized water as a solvent, and the pH value is 7.